Cytochrome P-450 3A13 and endothelin jointly mediate ductus arteriosus constriction to oxygen in mice

Abstract

The fetal ductus arteriosus (DA) contracts to oxygen, and this feature, maturing through gestation, is considered important for its closure at birth. We have previously obtained evidence of the involvement of cytochrome P-450, possibly of the 3A subfamily (CYP3A), in oxygen sensing and have also identified endothelin (ET)-1 as the attendant effector for the contraction. Here, we examined comparatively wild-type (WT) and CYP3A-null (Cyp3a(-/-)) mice for direct validation of this concept. We found that the CYP3A subfamily is represented only by CYP3A13 in the WT DA. CYP3A13 was also detected in the DA by immunofluorescence microscopy, being primarily colocalized with the endoplasmic reticulum in both endothelial and muscle cells. However, a distinct signal was also evident in the plasma membrane. Isolated DAs from term WT animals developed a sustained contraction to oxygen with transient contractions superimposed. Conversely, no tonic response occurred in Cyp3a(-/-) DAs, whereas the phasic response persisted unabated. Oxygen did not contract the preterm WT DA but caused a full-fledged contraction after retinoic acid (RA) treatment. RA also promoted an oxygen contraction in the Cyp3a(-/-) DA. However, responses of RA-treated WT and Cyp3a(-/-) mice differed in that only the former abated with ET-1 suppression. This implies the existence of an alternative target for RA responsible for the oxygen-induced contraction in the absence of CYP3A13. In vivo, the DA was constricted in WT and Cyp3a(-/-) newborns, although with a tendency to be less narrowed in the mutant. We conclude that oxygen acts primarily through the complex CYP3A13 (sensor)/ET-1 (effector) and, in an accessory way, directly onto ET-1. However, even in the absence of CYP3A13, the DA may close postnatally thanks to the contribution of ET-1 and the likely involvement of compensating mechanism(s) identifiable with an alternative oxygen-sensing system and/or the withdrawal of relaxing influence(s) operating prenatally.

Oxygen Sensing in the Ductus Arteriosus: Endothelin Still a Player

“…Collectively, then, the data purportedly contradicting an oxygen-sensing function for endothelin are not as firm as being portrayed by Hong et al 1 In the end, I believe it is intrinsically unrewarding to oppose 1 scheme for oxygen sensing to the other, and an effort should instead be made to determine how the 2 mechanisms may eventually dovetail in promoting ductus closure. This is our position 2 and, in fact, while validating the endothelin function, we have found evidence of an alternative, hitherto uncharacterized, oxygen-sensing mechanism 9 that could well represent the mitochondrial function being investigated by Hong et al…”

Functional Closure of the Ductus Arteriosus at Birth: Evidence against an Intermediary Role of Angiotensin II

“…Krebs medium was prepared as previously described [10] and was bubbled with gas mixtures containing 2.5 or 30% O 2 (plus 5% CO 2 and balance N 2 ) to mimic, respectively, the fetal and neonatal condition through the collection of ductus preparations (see below). Olmesartan medoxomil (courtesy of Daiichi Sankyo for part of our requirement; Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and PD123319 (Sigma-Aldrich, St. Louis, Mo., USA) were used as antagonists of AT 1 R and AT 2 R, respectively.…”

“…Ductus caliber in both fetus and newborn was assessed with the whole-body freezing technique as previously described [10]. …”

“…Ductus preparations were collected from term fetuses and 3-hour-old newborns, both olmesartan-treated and untreated, and were then processed according to a published protocol [10]. Briefly, specimens were pooled (12-27 preparations per group; three independent tests per gene) and total RNA was obtained using Tripure isolation reagent (Roche, Indianapolis, Ind., USA).…”

Therapeutic manipulation of the ductus arteriosus: current options and future prospects

“…Peculiar in many ways, the sensor recalls in its operation a conventional receptor, and this feature could provide means for a targeted manipulation. Specifically, it shows a selective distribution, with only few sites sharing such property with the ductus 24 ; a developmental profile which is independent from the maturation of the contractile function 25,26 ; desensitization upon repeated or extended exposure to the agent 27 ; and an accelerated expression upon treatment with certain compounds, particularly retinoic acid.…”